1. Field of the Invention
The invention generally relates to the enhancement of the delivery of and the in vivo nuclease resistance of antisense oligodeoxynucleotides (ODNs). In particular, the invention provides high affinity DNA binding platinum compounds which bind antisense oligodeoxynucleotides, facilitate their delivery into the cell, and augment their in vivo nuclease resistance. Additionally, the invention provides a novel method for treating cancer.
2. Background of the Invention
Antisense technology is a rapidly developing field, with many compounds being evaluated in preclinical and clinical trials as therapeutic agents in cancer, viral infections, genetic disorders and as tools for functional genomic studies. The basic goal of antisense technology is to inhibit protein expression by the binding of small molecules to single-stranded messenger RNA. Protein expression is inhibited either by physically blocking the translational machinery or by inducing RNase H-mediated RNA degradation.
An ODN is a short (about 7-25 nucleotides) sequence of single-stranded DNA which is complementary to the mRNA which is to be inhibited. Once introduced into the cell, the ODN can bind to the mRNA and prevent its translation. However, the ODN must 1) enter the cell in sufficient quantities and 2) remain stable until bound to the mRNA.(for reviews see Crooke, 1998; Branch, 1998; and Lavrovsky, 1997).
Some of the possible antisense molecules in development include synthetic oligonucleotides (typically modified in the phosphate backbone, the sugar moiety, and/or the nucleobase) (Schmajuk, 1999; Crooke, 1997; Monia, 1997; Flanagan, Wolf et al, 1999; Flanagan, Wagner et al, 1999), morpholino oligonucleotides (Summerton and Weller, 1997), chemically conjugated oligonucleotides (Spiller et al., 1998), peptide nucleic acids (Good and Nielsen, 1997; Nielsen, 1999), and plasmid-derived RNA (Weiss, 1999; Inouye et al., 1997). Also under development are antisense molecules which use alternate strategies for degrading the mRNA-ribozyme-mediated cleavage of the mRNA (Arndt and Rank, 1997; Rossi, 1997), trans-splicing ribozymes (Kohler, 1999), oligonucleotide-based external guide sequences (EGSs) which are substrates for Rnase P (Guerrier-Takada et al., 1995; Ma, 1998), and 2',5'-oligoadenylate-chimeric molecules which are substrates for RNase L (Cramer, 1999; Verheijen, 1999; Xiao et al., 1998).
The initial use of phosphodiester oligonucleotides revealed very poor penetration into the cell and a very short half-life in sera, on the order of 15-60 minutes (Crooke, 1992). The first generation of modified oligonucleotides were created by the incorporation of a phosphorothioate backbone. These modified oligonucleotides exhibited enhanced cellular stability of the oligonucleotide (the cellular lifetime increased to 24-48 hours) and were substrates for RNase H. However, DNA-RNA duplex stability was lowered, and toxic side effects were created as a result of metabolites and non-specific binding to proteins. Pharmacokinetic properties, tissue distribution, and in vivo stability data are now becoming available for these first generation antisense molecules. So far, the data indicate the need for improved modifications that will increase potency and decrease cytotoxicity (Agrawal, 1996; Juliano, 1999).
Chemistries that modify the sugar rather than the backbone of the oligonucleotide, such as 2'-O-methyl and 2'-O-methoxyethyl analogs (McKay et al., 1996; Monia, 1997; Monia et al., 1993) are the second generation of antisense oligonucleotides. These modifications increase DNA-RNA duplex stability, show partial resistance to nucleases, and have a lower toxicity than the corresponding phosphorothioate oligonucleotides. The major drawback of these modifications is the inhibition of the RNase H mechanism which is necessary for degrading the targeted mRNA; there is no antisense effect because the mRNA can be translated into functional protein. Currently, mixed-backbone oligonucleotides, chimeric oligomers that incorporate both phosphorothioates and 2'-modified sugars, have become the method of choice. These molecules comprise a mixture of chemistries that attempt to optimize stability, specificity, nuclease resistance, and are still substrates for RNase H (Agrawal and Zhang, 1997; Crooke, 1997).
While engineering newer and better antisense oligomers remains important, focus has shifted to optimizing delivery and cellular localization of these molecules. Antisense molecules can enter the cell by passive diffusion or by receptor-mediated endocytosis, although the mechanism of oligonucleotide release from the endosome is not well understood. However, neither mechanism is efficient, resulting in little or no antisense molecule actually getting to its target. For example, leukemia MOLT-3 cells incubated with an unmodified phosphodiester oligonucleotide showed no cellular uptake and a fully-modified phosphorothioate oligonucleotide showed only 0.3% of the initial concentration of oligonucleotide incorporated into cells (Thierry and Dritschilo, 1992). Typically, high concentrations (.mu.M) of antisense oligonucleotides are used to increase the intracellular concentration, which potentially leads to increased cytotoxicity.
Cellular uptake of antisense oligonucleotides in vitro can be dramatically improved through the use of uptake mediators such as cationic lipids, which capitalize on the electrostatic interaction between the positively-charged compound and the negatively-charged ODN for delivering the molecule to the nucleus (Hope et al., 1998). Many commercial cationic lipids exist; however, they have a tendency to aggregate and can be toxic at high concentrations. In addition, the ratio of transfection agent to oligomer is highly empirical and needs to be optimized for every cell line. An alternate method for packaging the antisense oligomer for cellular uptake is liposome encapsulation. Typically, these formulations involve a combination of cationic polymers, such as phosphotidylcholine or phosphotidylethanolamine, and lipophilic molecules (Gokhale et al., 1997).
Other novel transfection agents currently under development which take advantage of electrostatic interactions between the compound and the ODN include a molecular umbrella, which assists the delivery of oligomers through the lipid bilayer (Janout et al., 1997), water-soluble cationic porphyrins (Benimetskaya, 1998; Takle et al., 1997), and Starburst polyamidoamine dendrimers (Helin, 1999; Bielinska et al., 1996). Also under development are antisense molecules that are chemically conjugated to delivery enhancers such as lipophilic conjugates (Rump et al., 1998; Spiller et al., 1998), and fusion peptides containing both hydrophobic and hydrophilic sequences (Chaloin et al., 1998).
While much research has been directed toward the discovery of new modes of delivery of antisense ODNs, so far none have succeeded in surmounting the challenges of combining both efficient delivery of the ODN to the cell, and resistance to endogenous nucleases after delivery. Thus, there remains a need for new, simple and innovative methods for enhancing cellular uptake of and increasing the in vivo stability of antisense molecules.